A minor component of the binding of [3H]guanyl-5'-yl imidodiphosphate to cardiac membranes associated with the activation of adenylate cyclase.
Incubation of a preparation of cardiac plasma membranes with 20 microM (--)-isoproterenol and 1 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) results in prolonged and maximal activation of adenylate cyclase and prolonged binding of [3H]Gpp(NH)p to the membranes. The total number of [3H]Gpp(NH)p sites must exceed those required for enzyme activation (N-sites) since the rate of binding is much slower than the rate of activation. Attempts to measure the amount of [3H]Gpp(NH)p released from N-sites during incubation of membranes with isoproterenol and GTP were frustrated by limited (about 20%) reversal of binding and of enzyme activation. However, the amount of [3H]-Gpp(NH)p bound to membranes could be reduced by preincubating them in a concentration of App(NH)p (0.1-0.5 mM) which does not interact with the N-site and most of the [3H]Gpp(NH)p subsequently bound could be removed by further incubation of the membranes with EDTA, propranolol, and Gpp(NH)p. Under these conditions, full enzyme activation was retained and was associated with residual bound [3H]Gpp(NH)p of about 0.3 pmol/mg of protein. This amount equals the concentration of beta-adrenoreceptors; and the rate and Kd (0.22 microM) for the binding residual [3H]-Gpp(NH)p approximate the rate and Ka for enzyme activation. The rate of enzyme activation and [3H]-Gpp(NH)p binding to the residual component was increased in the presence of isoproterenol. Competition binding studies for the residual component revealed that Gpp(NH)p congruent to GTP greater than GDP greater than GMP. The results suggest that residual [3H]Gpp(NH)p binding is to the adenylate cyclase-coupled guanine nucleotide binding protein.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004492:Edetic Acid / Q000494:pharmacology*; D004789:Enzyme Activation; D005260:Female; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding
D000266:Adenylyl Imidodiphosphate; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D000262:Adenylyl Cyclases